•öáñÓ³ñ³ñ³Ï³Ý ¨ ï»ë³Ï³Ý Ñá¹í³ÍÝ»ñ • Экспериментальные и теоретические статьи•
•Experimental and Theoretical articles•
Biolog. Journal of Armenia, 1 (61), 2009
PRODUCTION OF BIOMASS OF PHOTOSYNTHETIC
BACTERIA ON BASE OF STOCK-BREEDING AND
POULTRY WASTES
A.Kh. PARONYAN, A.V. GASPARYAN
Centre of Microbiology and Microbial Depository (CMMD),
NAS of Armenia, Abovyan city
On base of stock-breeding and poultry wastes the cheap nutrient media for
cultivation and production of biomass of phototrophic bacteria have been
developed. Cultivation of purple nonsulfur bacteria Rhodobacter sphaeroides
together with Bacillus megaterium in mixed culture has been succeeded in high
biomass yield. The obtained biomass was distinguished with rich contents of
biologically active substances.
Photosynthetic bacteria – biomass – mixed culture
Անասնապահական և թռչնաբուծական թափոնների հիման վրա մշակվել
են էժան սննդամիջավայրեր ֆոտոտրոֆ բակտերիաների աճեցման և
կենսազանգվածի ստացման համար։ Կիրառելով խառը կուլտուրաների՝
Rhodobacter sphaeroides ոչ ծծմբային ծիրանագույն բակտերիաների և Bacillus
megaterium□ի համատեղ աճեցումը, հաջողվել է զգալի բարձրացնել
կենսազանգվածի ելքը։ Ստացված կենսազանգվածը առանձնանում է
կենսաբանական ակտիվ միացությունների հարուստ պարունակությամբ։
Ֆոտոսինթետիկ բակտերիաներ □ կենսազանգված □ խառը կուլտուրա
Разработаны дешевые питательные среды на основе животноводческих и
птицеводческих отходов для выращивания и получения биомассы фототрофных бактерий. С использованием смешанного культивирования несерных
пурпурных бактерий Rhodobacter sphaeroides и Bacillus megaterium удалось
заметно увеличить выход биомассы. Полученная биомасса отличается богатым содержанием биологически активных соединений.
Фотосинтетические бактерии – биомасса – смешанная культура
The abilities of photosynthetic bacteria to assimilate carbon dioxide, to fix
molecular nitrogen possessing with photosynthetic metabolism, as well as to grow
on different wastes permit to use phototrophs in biotechnology [7,11]. The
phototrophic bacteria are effective producers of biologically active substances, such
as carotinoides, organic acids, moreover the biomass of phototrophs contains
valuable substances and has practical importance as feed protein [2, 10].
5
A.Kh. PARONYAN, A.V. GASPARYAN
Application of phototrophs in Armenia has high perspectives since this group
of microorganisms are widely distributed, especially, in mineral springs, lakes,
natural reservoirs, and can be grown from early spring till late autumn [6]. The
literature data on cultivation and production of biomass of phototrophic bacteria on
different agricultural wastes as nutrient media are less known.
The present work reports the results of investigation on production of biomass
of phototrophic bacteria using as nutrient media the agricultural wastes such as
stock-breeding manure and poultry dung.
Materials and methods. The strains of purple nonsulfur photosynthetic bacteria
Rhodobacter sphaeroides sp.D-8 and Rhodobacter sphaeroides sp. A-10 isolated from
mineral springs of Jermuk and Arzni as well as strain Bacillus megaterium INMIA
B-1502 have been studied. All strains of bacteria were deposited in Culture Collection of
the Centre of Microbiology and Microbial Depository (CMMD), NAS of Armenia.
Strains of phototrophic bacteria were grown on Ormerud's medium [9]. The strain
of Bacillus megaterium was grown on a medium containing fish hydrolysate [1].
The agricultural wastes such as stock-breeding manure and poultry dung were
periodically taken from stock-breeding and poultry farms of Abovyan region. The wastes
were dried at 60° for 15 days until containing 30 % of humidity in manure and 20% of
humidity in dung. The 100 g dry matter samples after grinding were dissolved in one
litre of tap water and were kept under room conditions periodically stirring for 4-6 hours.
After filtration the extracts obtained were used as a base of nutrient media for cultivation
of phototrophs. The initial pH was nearly 6.5-6.9. The chemical analysis of extracts
showed that they contain ions of HCO3 - 1321.0-1858. 5 mg/l, carbohydrates (glucose,
sucrose) - 0.08-0.86 %, salts of organic acids which could satisfy the growth
requirements of purple nonsulfur bacteria.
The phototrophs were grown at 30-32° with 2500–3000 lux illuminating and
under different conditions of aeration. The inoculum was added in amount of 5 %
(v/v) in the nutrient media. The growth of cultures was monitored spectrophotometrically determining the optical density of cell suspension at wave length 660
nm and by determining the dry weight of bacterial biomass. Microscopic control was
performed using microscope MBB-1A and a KF-4 phase contrast device. Absorption
spectra of whole cells were measured on “Specord” UV-Vis spectrophotometer. The
phototrophs were grown for 5-8 days until the considerable de-crease of growth. The
content of wet protein was determined by nitrogen method. The amino acids composition
was determined on amino acid analyzer AAA-339. The vitamins were determined by
Odincova’s method [8 ], vitamin B12 – by Kuceva’s method [5].Three-day-old cultures
of phototrophic bacteria grown ana-erobically under 2500-3000 lux illumination and
one-day-old culture of B. mega-terium grown on a flask-shaker were used as an
inoculum for mixed cultivation with optimal combination 2:1 (v/v) of phototrophic and
heterotrophic cultures.
Statistical analysis was based on date from experiments of ten replicates.
Results and discussion. The yield of biomass is few on clean extracts. It is
explained by the high contents of calcium salts in extracts which affects on
penetration of light increasing the optical density of the media. Good results of
the growth were mentioned when the extracts were diluted with tap water in
ratio 1:1(v/v). The accumulation of biomass in considerably increased when
small amount of yeast extract (0.02%) is added in different media as a growth
stimulator. The results of composition and content of amino acids (Table l), total
nitrogen and protein (Table 2), valuable vitamins (Table 3) and carotenoids
( Fig. 1) of obtained biomass are represented respectively on tables.
6
PRODUCTION OF BIOMASS OF PHOTOSYNTHETIC BACTERIA ON BASE OF ...
Table 1. Amino acid composition of biomass of Rh. sphaeroides sp.
D-8 grown on different media (g/100g dry weight of biomass)
Amino acids
DL-Alanine
L-Arginine
DL-Aspartic acid
Glycine
L-Glutamic acid
L-Histidine
L-Isoleucine
DL-Leucine
DL-Lysine
DL-Methionine
DL-Phenylalanine
L-Proline
DL-Serine
DL-Threonine
DL-Tryptophane
Dl-Tyrosine
DL-Valine
Ormerud’s
medium
(control)
4.57
3.2
4.34
2.38
5.43
1.1
2.42
3.15
2.72
1.08
1.99
2.6
1.58
2.83
+
1.48
3.43
Medium with
stock-breeding
manure
4.55
3.2
4.35
2.18
5.43
1.1
2.25
2.88
2.68
0.85
1.9
2.5
1.58
2.8
+
1.3
3.3
Medium with
poultry
dung
4.3
3.12
4.05
2.0
5.33
1.05
2.2
2.72
2.68
0.64
1.69
2.16
1.58
2.68
+
1.07
3.11
Table 2. Contents of total nitrogen and protein in biomass of
phototrophic bacteria grown on different media
Media
1)Total nitrogen, 1)Protein, 2)Total nitrogen, 2)Protein,
%
%
%
%
Ormerud’s
9.4
59
8.67
54.1
medium (control)
Medium with
9.25
58.6
8.45
53.7
stock-breeding
manure
Medium with
9.2
58.2
8.22
53.3
poultry dung
Legend: 1)- Rh. sphaeroides sp. D-8, 2) – Rh. sphaeroides sp. A-10.
Table 3. Contents of vitamins in biomass of Rh. sphaeroides sp. D-8
Amino acids
Ormerud’s
Medium with stockmedium
breeding manure
Biotin
0.42
0.39
Cyanocobalamin
513
503
Niacin
415
398
Pantothenic acid
2.5
2.3
Pyredoxin
2.6
2.6
Thiamin
6.03
5.8
Legend: Vitamins content, µg/g dry weight.
Medium with
poultry dung
0.37
487
389
2.3
2.17
5.35
7
A.Kh. PARONYAN, A.V. GASPARYAN
The results of composition and content of amino acids, total nitrogen and
protein, valuable vitamins in biomass obtained on media with waste extracts
didn’t give up and in some cases exceeded the same results on the control
Ormerud's medium (Tables 1, 2, 3).
It is known that the organic compounds of carbon serve as a source of
carbon and electron donor for purple nonsulfur bacteria. The less of car-bon
source media affects not only on growth of the cultures but also on biosyntheses
of carotinoides.
The absorption spectra showed that the synthesized carotinoides on
medium with stock-breeding manure by their quantitative and qualitative
characteristics are similar to same results obtained on control Ormerud's
medium (Fig. 1).
Fig. 1. Absorption spectra of whole cells of Rh. sphaeroides sp. D-8 grown in different
media: 1. Ormerud’s medium, 2. Medium with stock-breeding manure
The results showed that the extracts of poultry dung were poor of so-me
elements in comparison with the stock-breeding manure. The addition of 0.3%
glucose to poultry dung stimulates the growth of bacteria up to 3.3-3.8g/l. It is
more effective to use molasses in concentration of 0.25% in media with the
extracts of poultry dung.
It is known that during the mixed cultivation of different microor-ganisms
the constant balance or stimulation of the growth of single or mixed cultures of
microorganisms is observed. Physiological properties of microorganisms, the
composition of media and the conditions of cultivation have great role in this
process [3]. The data on cultivation statistics of mixed cultures under different conditions
confirm the great influence on the growth of monocultures and mixed cultures [4].
In our experiments we have used the method of mixed cultivation for
production of high level of biomass of phototrophic bacteria on base of wastes.
As heterotrophic microorganism B. megaterium was used.
The purple nonsulfur bacteria are facultative anaerobes though they grow
also well under conditions of weak aeration in dark. In association of facultative
anaerobic phototrophic bacteria and aerobic B. megaterium es-sentially the
partners are differed with their requirements to oxygen.
The results on mixed cultivation of phototrophic bacteria and B. megaterium are represented in Table 4.
8
PRODUCTION OF BIOMASS OF PHOTOSYNTHETIC BACTERIA ON BASE OF ...
In our experiments the active growth of B. megaterium was observed
under aerobic conditions using both monoculture and mixed culture. Microairation in mixed culture increased the growth of phototrophs and decreased
the growth of heterotrophic culture but the heterotrophic culture in this case had
rather stimulating influence on growth of phototrophs. In case of anaerobic
conditions the growth of phototrophic bacteria was high in monoculture but the
single cells of B. megaterium were observed in monoculture. In mixed culture
the growth of phototrophic bacteria under anaerobic conditions was high in
comparison with monoculture. In this case the stimulation effect of B.
megaterium even with weak growth was available (Table 4).
Table 4. Influence of aeration on the biomass yield during mixed cultivation of
phototrophic bacteria and B. megaterium on medium with stock-breeding manure
extracts (g/l dry weight of biomass)
Cultures
Rh. sphaeroides, sp. D-8
Rh. sphaeroides, sp. A-10
B. megaterium
Rh. sphaeroides, sp. D-8 +
B. megaterium
Rh. sphaeroides, sp. A-10 +
B. megaterium
Aerobic
(dark)
0.43
0.54
2.2
Microaerophilic
(light)
3.36
3.85
1.9
Anaerobic
(light)
3.06
3.7
0.048
2.41
4.8
4.24
2.66
5.03
4.94
The analysis of the growth of single culture-components showed that the
mixed growth of cultures is based on symbiotic interaction as commensalism.
The commensal relations among phototrophic and heterotrophic bacteria have
their best effect under microaerophilic conditions. The stimulating influence of
heterotrophic bacterial culture was the highest when the correlation of
phototrophic and heterotrophic bacteria was in ratio 2:1(v/v) in inoculum. In
association of phototrophic and heterotrophic bacteria the yield of biomass could
be increased about 33-42% in comparison with monoculture. Microscopic
examination conformed that the biomass obtained was mainly consisted of
phototrophic bacteria.
Thus, the cheap nutrient media on the base of agricultural wastes such as
stock-breeding manure and poultry dung have been developed for production of
biomass of phototrophic bacteria. The highest yield of biomass was observed by
mixed cultivation under microaerophilic conditions of growth. Besides being
rich feed product the biomass can be also used as a source of valuable
biologically active substances and can be applied as fertilizer in agriculture.
1.
2.
3.
4.
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Received 24.12.2008
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